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Biosynthesis of Histone Messenger RNA Employs a Specific 3' End

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Biosynthesis of Histone Messenger RNA Employs a Specific 3' End RESEARCH ARTICLE Biosynthesis of histone messenger RNA employs a specific 3’ end endonuclease Ilaria Pettinati1, Pawel Grzechnik2, Claudia Ribeiro de Almeida3, Jurgen Brem1, Michael A McDonough1, Somdutta Dhir3, Nick J Proudfoot3*, Christopher J Schofield1* 1Department of Chemistry, University of Oxford, Oxford, United Kingdom; 2School of Biosciences, University of Birmingham, Birmingham, United Kingdom; 3Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom Abstract Replication-dependent (RD) core histone mRNA produced during S-phase is the only known metazoan protein-coding mRNA presenting a 3’ stem-loop instead of the otherwise universal polyA tail. A metallo b-lactamase (MBL) fold enzyme, cleavage and polyadenylation specificity factor 73 (CPSF73), is proposed to be the sole endonuclease responsible for 3’ end processing of both mRNA classes. We report cellular, genetic, biochemical, substrate selectivity, and crystallographic studies providing evidence that an additional endoribonuclease, MBL domain containing protein 1 (MBLAC1), is selective for 3’ processing of RD histone pre-mRNA during the S-phase of the cell cycle. Depletion of MBLAC1 in cells significantly affects cell cycle progression thus identifying MBLAC1 as a new type of S-phase-specific cancer target. DOI: https://doi.org/10.7554/eLife.39865.001 Introduction During S-phase of the cell cycle, production of the core histone proteins (H2A, H2B, H3, and H4) is *For correspondence: coordinated with DNA replication (Harris et al., 1991; Ewen, 2000). Metazoan mRNAs encoding [email protected] (NJP); for the ‘replication-dependent’ (RD) core histones lack the normal polyA tail formed by 3’ end hydro- [email protected]. lysis of pre-mRNA followed by polyadenylation (Proudfoot, 2011). Instead, they undergo endonu- ac.uk (CJS) cleolytic cleavage at the 3’ side of an RNA hairpin (stem loop) producing mRNA with a 3´stem loop (SL), which is exported from the nucleus for use in translation (Marzluff et al., 2008; Marzluff and Competing interest: See Koreski, 2017). By contrast, the pre-mRNA of replication-independent histone variants are normally page 23 polyadenylated and constitutively expressed during the cell cycle (Marzluff et al., 2002; Funding: See page 23 Wagner et al., 2007). Received: 05 July 2018 A single endonuclease, cleavage and polyadenylation specificity factor 73 (CPSF73), is proposed Accepted: 30 November 2018 to be responsible for the hydrolysis of both RD histone pre-mRNA (SL) and normal protein-coding Published: 03 December 2018 pre-mRNA (polyA) (Dominski et al., 2005; Mandel et al., 2006; Kolev et al., 2008; Sullivan et al., 2009b; Dominski, 2010). Although maturation of both classes of RNAs requires a hydrolytic reac- Reviewing editor: Torben Heick Jensen, Aarhus University, tion, different macromolecular complexes are recruited to the different pre-mRNA classes (SL or Denmark polyA) (Kolev et al., 2008; Sullivan et al., 2009b). Specific factors involved in RD histone pre-mRNA maturation are the stem loop binding protein (SLBP), the FLICE-associated huge protein (FLASH), Copyright Pettinati et al. This and the U7 small nuclear ribonucleoprotein (U7snRNP) that binds to a histone downstream element article is distributed under the (HDE) (Dominski et al., 2005; Kolev et al., 2008; Sullivan et al., 2009b). Defective 3’ end process- terms of the Creative Commons Attribution License, which ing of RD histone pre-mRNA, caused by depletion of factors including CPSF73, SLBP, Sm-like pro- permits unrestricted use and tein 11 (Lsm11), CstF64 or FLASH, results in the generation of extended transcripts downstream of redistribution provided that the the HDE sequence (Sullivan et al., 2009a; Romeo et al., 2014; Sullivan et al., 2009b). In Drosophila original author and source are melanogaster and humans, misprocessed RD histone pre-mRNA has been observed to undergo pol- credited. yadenylation involving utilization of a secondary polyadenylation signal sequence located Pettinati et al. eLife 2018;7:e39865. DOI: https://doi.org/10.7554/eLife.39865 1 of 26 Research article Cell Biology Chromosomes and Gene Expression downstream of the HDE (Sullivan et al., 2009b; Romeo et al., 2014; Kari et al., 2013). Depletion of factors belonging to the 5’ cap-binding complex (CBC) (Hallais et al., 2013, Narita et al., 2007; Gruber et al., 2012), or to the cleavage factor II (CF IIm), which is normally involved in 3’ end proc- essing of normal protein-coding pre-mRNA (polyA) (Hallais et al., 2013; de Vries et al., 2000), also results in extended RD histone pre-mRNA transcripts (Hallais et al., 2013). These observations sug- gest a complex and dynamic relationship between the factors involved in the different stages of the RD histone pre-mRNA transcription process, which may involve participation of factors normally belonging to the polyA mRNA processing machinery. Important cancer medicines, including histone deacetylase and cyclin-dependent kinase inhibi- tors, target proteins involved in the S-phase (Newbold et al., 2016; Falkenberg and Johnstone, 2014). In work aimed at identifying potential new S-phase cancer targets, we considered known and potential roles of MBL-fold proteins involved in nucleic acid hydrolysis (Dominski, 2007; Pettinati et al., 2016; Daiyasu et al., 2001). In addition, to the role of CPSF73, and the likely pseudo-enzyme CPSF100, in pre-mRNA processing (Dominski et al., 2005; Mandel et al., 2006), MBL-fold nucleases are involved in DNA repair (SNM1A-C nucleases) (Yan et al., 2010), snRNA processing (INTS9 and INTS11), and tRNA processing (ELAC 1 and 2) (Skaar et al., 2015; Vogel et al., 2005). Whilst most of the ~18 human MBL-fold proteins have established functions (Pettinati et al., 2016), the functions of several are unassigned, including the MBL domain contain- ing protein 1 (MBLAC1). Here, we report evidence that MBLAC1 is a nuclease specific for cleavage of RD histone pre-mRNA. Crystallographic and biochemical studies show that MBLAC1 has an over- all MBL fold and di-zinc ion containing active site related to that of CPSF73, but which has distinctive structural features involving active site flanking loops and the absence of the b-CASP domain, which is only present in CPSF73. MBLAC1 depletion from cells leads to the production of unprocessed RD histone pre-mRNA due to inefficient 3’ end processing. The consequent depletion of core histone proteins correlates with a cell cycle defect due to a delay in entering/progressing through S-phase. Results MBLAC1 structure reveals similarity with MBL-fold nucleases On the basis of sequence similarity studies MBLAC1 has been assigned as an RNAse Z and glyoxa- lase II subfamily enzyme (Ridderstro¨m et al., 1996; Sievers et al., 2011)(Figure 1—figure supple- ment 1A). However, we found that recombinant MBLAC1 prepared from E. coli has only low, likely non-specific, glyoxalase activity as observed for other hMBL-fold proteins belonging to the same subfamily (Shen et al., 2011). To investigate its function, we solved a crystal structure of MBLAC1 (1.8 A˚ resolution, space group P1) (Table 1). The structure reveals a stereotypical abba MBL- fold (Carfi et al., 1995) with two central mixed b-sheets (I and II), comprised of 8 and 5 strands respec- tively, surrounded by helices (Figure 1A). In b-sheet I, b-strands 1, 2, 5–6 and 8–10 are anti-parallel, with b-strands 6–8 being parallel; b-strands 3 and 4 are part of a loop region and are aligned anti- parallel to each other and parallel to b-strands 2 and 5, respectively. In b-sheet II, b-strands 11, 12, 13 and 14 are anti-parallel, and b-strands 14 and 15 are parallel (Figure 1A). MBLAC1 has four of the five characteristic MBL-metal-binding motifs (Pettinati et al., 2016), His116, His118 Asp120 and His121 (motif II), His196 (motif III), Asp221 (motif IV) and His263 (motif V) (Figure 1B) (using BBL numbering) (Galleni et al., 2001) with two waters completing metal coordination. In the structure of recombinant MBLAC1 produced in E. coli, the active site was refined with two iron ions present (Figure 1B). However, MBLAC1 produced in HEK293 cells preferentially binds zinc ions (Figure 1C). Four MBLAC1 molecules (chains A-D) are present in the crystallographic asymmetric unit; analysis of interactions at the crystallographically observed monomer interfaces (Krissinel and Henrick, 2007) identified interactions between chains A-B and C-D (Figure 1D) possibly reflecting dimeric MBLAC1 in solution (Figure 1E and F). The metal containing active site is adjacent to the dimer interface (Figure 1D), rationalizing reduced dimerization as manifested by metal ligand substitution or metal removal (Figure 1E–G). Comparison of the MBLAC1 structure with those of other MBL-fold proteins reveals that it is indeed part of the RNAse Z/glyoxalase II MBL structural subfamily (Figure 1H; Figure 1—figure sup- plement 1B–C). However, although there is low overall sequence similarity (27%), the overall MBLAC1 fold is structurally similar to the human endoribonuclease b-lactamase-like-protein 2 Pettinati et al. eLife 2018;7:e39865. DOI: https://doi.org/10.7554/eLife.39865 2 of 26 Research article Cell Biology Chromosomes and Gene Expression Table 1. Crystallographic data and refinement statistics PDB ID 4V0H. Native HSE (PDB ID: 4V0H) Data collection Space group P1 Cell dimensions a, b, c (A˚ ) 62.95, 67.13, 67.90 a, b, g (˚) 109.31, 105.40, 90.17 Resolution (A˚ ) 45.96–1.79 (1.84–1.79 A˚ )* † Rmerge 0.10 (0.81) I/s(I) 12.4 (2.6) Completeness (%) 95.5 (92.6) Redundancy 6.9 (6.7) Refinement Resolution (A˚ ) 45.95–1.79 No. reflections 90641 ‡ § Rwork /Rfree 0.182/0.211 No. atoms Protein 6235 Ligand/ion 38 Water 514 B factors Protein 27.27 Ligand/ion 44.40 Water 33.94 R.m.s. deviations Bond lengths (A˚ ) 0.01 Bond angles (˚) 1.37 * Values in parentheses are for highest-resolution shell.
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